1. Field of the Invention
The present invention relates to novel non-peptide mimetics of CAAX (C.dbd.Cys, A.dbd.Leu, Ile or Val, X.dbd.Met or Ser) which are useful as inhibitors of Ras farnesyltransferase (FTase).
Cysteine farnesylation of the carboxyl terminal tetrapeptides CAAX of the oncogene product Ras is required for its malignant transformation activity. As a consequence, farnesyltransferase (FTase), the enzyme responsible for the essential lipid modification, has become one of the most sought after targets for anticancer drug development. Previously, proposed peptide mimics have shown potent inhibition of FTase in vitro but have certain undesirable peptide features that hamper their use in vivo. The invention is concerned with non-peptide mimetics of CAAX which are free from peptide amide groups and other peptidic features which are effective and selective for FTase inhibition both in vitro and in vivo.
2. Background Information
Ras is a small guanine nucleotide binding GTPase that transduces biological information from the cell surface to the nucleus (1). Its ability to transfer growth signals from receptor tyrosine kinases to a mitogen activated protein (MAP) kinase cascade puts it in the heart of signaling pathways that cause proliferation in normal cells and uncontrolled growth in cancer cells (2). Indeed, mutations that lock ras in its active, GTP-bound state lead to malignant transformation and are among the most frequently identified mutations in human cancers (1). For example, 50% of colorectal and 95% of pancreatic human cancers have activated ras oncogenes.
Over the last decade, several strategies have been investigated, with only moderate success, to disrupt Ras function and hence, to inhibit the growth of tumors with activated ras oncogenes. The search has recently intensified with the discovery that Ras requires lipid modification with a farnesyl group for localization to the plasma membrane where it plays a pivotal role in growth signaling (3-9). Because farnesylation is required and sufficient for ras membrane association and transformation (10), the enzyme that catalyzes this lipid modification, farnesyltransferase (FTase), has become a major target for the design of novel anticancer agents (11, 12).
FTase is a .alpha. and .beta. heterodimer that transfers farnesyl from farnesylpyrophosphate, a cholesterol biosynthesis intermediate, to the cysteine of proteins containing the carboxyl terminal sequence CAAX (where C is cysteine, A is an aliphatic amino acid and X is any amino acid except Leu or Ile) (13, 14). A closely related prenyltransferase, geranylgeranyltransferase I (GGTase I) catalyzes cysteine geranylgeranylation of proteins ending in CAAX where X-Leu or Ile (15, 16). In contrast to FTase and GGTase I, a third prenyltransferase, GGTase II, does not recognize CAAX sequences but rather geranylgeranylates proteins ending in CC or CXC sequences (15, 16). Prenylation of CAAX sequences by FTase and GGTase I is followed by proteolysis of the tripeptide AAX and carboxymethylation of the resulting prenylated cysteine, whereas GGTase II-catalyzed reactions are not followed by further posttranslational modifications. Since the number of geranylgeranylated proteins in the cell far exceeds that of farnesylated proteins (15, 16), it is desirable that potential anticancer agents which act by targeting farnysylation be highly selective for FTase over GGTase I to minimize side effects.
Developing Ras CAAX tetrapeptide mimics as anticancer drugs has been prompted by the observation that FTase recognizes and farnesylates CAAX peptides which were also found to be potent competitive inhibitors of the enzyme (IC.sub.50 s=50-200 nM) (13, 17-22). However, because of their peptidic nature, CAAX peptides do not inhibit Ras processing in whole cells. To enhance their poor cellular uptake and decrease their sensitivity to cellular proteases, it has been proposed to make CAAX pseudopeptides (23-25). Reduction of the amino terminal and central amide bonds of CAAX, and neutralization of the free carboxylate resulted in greater activity in whole cells (23-25)
Although the FTase inhibitors discussed above are potent inhibitors in vitro, they still retain several peptidic features which leave the proposed inhibitors vulnerable to proteolytic degradation in cells. With this in mind, it has been proposed in U.S. Pat. No. 5,602,098 to modify prior CAAX tetrapeptide mimics by, for example, replacing the peptide portion VI in CVIM (SEQ ID NO.1), the carboxyl terminal of K.sub.B -Ras, by a hydrophobic spacer such as 4-aminobenzoic acid (4-ABA) to link Cys to Met (i.e., Cys-4ABA-Met) (26-28). As an alternative, James et al (29) used a benzodiazepine group between cysteine and methionine. The latter suggested that the best disruption of FTase activity could be achieved when the inhibitors take up a .beta. turn conformation that brings in close proximity the cysteine thiol and the methionine carboxylate to form a bidentate complex with a putative Zn.sup.++ ion in the enzyme active site (29). However, conformational analysis of the inhibitor Cys-4ABA-Met described in U.S. Pat. No. 5,602,098 has shown that it could not take up a .beta.-turn, arguing against this structural feature as a target for future FTase inhibitor design (27).
The compounds of U.S. Pat. No. 5,602,098 represent a significant improvement over prior FTase inhibitors. However, it is believed that even better results could be obtained if the amide bond or bonds or other peptidic features could be eliminated or at least reduced even further.